Gentile Francesco Ficetola

Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.

An In silico approach for the evaluation of DNA barcodes

BackgroundDNA barcoding is a key tool for assessing biodiversity in both taxonomic and environmental studies. Essential features of barcodes include their applicability to a wide spectrum of taxa and their ability to identify even closely related species. Several DNA regions have been proposed as barcodes and the region selected strongly influences the output of a study. However, formal comparisons between barcodes remained limited until now. Here we present a standard method for evaluating barcode quality, based on the use of a new bioinformatic tool that performs in silico PCR over large databases. We illustrate this approach by comparing the taxonomic coverage and the resolution of several DNA regions already proposed for the barcoding of vertebrates. To assess the relationship between in silico and in vitro PCR, we also developed specific primers amplifying different species of Felidae, and we tested them using both kinds of PCRResultsTests on specific primers confirmed the correspondence between in silico and in vitro PCR. Nevertheless, results of in silico and in vitro PCRs can be somehow different, also because tuning PCR conditions can increase the performance of primers with limited taxonomic coverage. The in silico evaluation of DNA barcodes showed a strong variation of taxonomic coverage (i.e., universality): barcodes based on highly degenerated primers and those corresponding to the conserved region of the Cyt-b showed the highest coverage. As expected, longer barcodes had a better resolution than shorter ones, which are however more convenient for ecological studies analysing environmental samples.ConclusionsIn silico PCR could be used to improve the performance of a study, by allowing the preliminary comparison of several DNA regions in order to identify the most appropriate barcode depending on the study aims.

Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data

Environmental DNA (eDNA) metabarcoding is increasingly used to study the present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of the presence/absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in the presence of observational errors can successfully estimate true prevalence, detection probability and false‐positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability and false‐positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases, collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies and could be explicitly estimated to improve the reliability of results.

Population genetics reveals origin and number of founders in a biological invasion

Propagule pressure is considered the main determinant of success of biological invasions: when a large number of individuals are introduced into an area, the species is more likely to establish and become invasive. Nevertheless, precise data on propagule pressure exist only for a small sample of invasive species, usually voluntarily introduced. We studied the invasion of the American bullfrog, Rana catesbeiana, into Europe, a species that is considered a major cause of decline for native amphibians. For this major invader with scarce historical data, we used population genetics data (a partial sequence of the mitochondrial cytochrome b gene) to infer the invasion history and to estimate the number of founders of non‐native populations. Based on differences between populations, at least six independent introductions from the native range occurred in Europe, followed by secondary translocations. Genetic diversity was strongly reduced in non‐native populations, indicating a very strong bottleneck during colonization. We used simulations to estimate the precise number of founders and found that most non‐native populations derive from less than six females. This capability of invasion from a very small number of propagules challenges usual management strategies; species with such ability should be identified at an early stage of introduction.

Influence of landscape elements in riparian buffers on the conservation of semiaquatic amphibians.

Studies on riparian buffers have usually focused on the amount of land needed as habitat for the terrestrial life stages of semiaquatic species. Nevertheless, the landscape surrounding wetlands is also important for other key processes, such as dispersal and the dynamics of metapopulations. Multiple elements that influence these processes should therefore be considered in the delineation of buffers. We analyzed landscape elements (forest cover, density of roads, and hydrographic network) in concentric buffers to evaluate the scale at which they influence stream amphibians in 77 distinct landscapes. To evaluate whether our results could be generalized to other contexts, we determined whether they were consistent across the study areas. Amphibians required buffers of 100-400 m of suitable terrestrial habitat, but interspecific differences in the amount of habitat were large. The presence of amphibians was related to roads and the hydrographic network at larger spatial scales (300-1500 m), which suggests that wider buffers are needed with these elements. This pattern probably arose because these elements influence dispersal and metapopulation persistence, processes that occur at large spatial scales. Furthermore, in some cases, analyses performed on different sets of landscapes provided different results, which suggests caution should be used when conservation recommendations are applied to disparate areas. Establishment of riparian buffers should not be focused only on riparian habitat, but should take a landscape perspective because semiaquatic species use multiple elements for different functions. This approach can be complex because different landscape elements require different spatial extents. Nevertheless, a shift of attention toward the management of different elements at multiple spatial scales is necessary for the long-term persistence of populations.

Trade-off between larval development rate and post-metamorphic traits in the frog Rana latastei

Development rate early in the ontogeny is believed to correlate positively with fitness. Geographic variation in intrinsic development rate suggests the existence of trade-offs between development rate and other fitness related traits. We investigated whether these trade-offs exist between intrinsic larval development rate and post-metamorphic traits in an organism with a complex life cycle. In laboratory, we measured if the tadpoles of the frog Rana latastei with fast intrinsic development rate have a suboptimal post-metamorphic morphology, by comparing froglets from five populations. Then, we evaluated the relationship between age at metamorphosis, hindlimb length and jumping performance for frogs grown in nature in two populations. Under laboratory conditions, froglets with fast intrinsic development had shorter absolute and shorter size-adjusted tibiofibulas. We observed a strong, positive relationship between tibiofibula length and jumping performance. In nature, froglets from the last metamorphosing population had longer absolute and size-adjusted tibiofibulas, and were able to jump further. The cost of fast development could be the shorter legs of early metamorphosing frogs, and their poor jumping performance. Thus, a fast intrinsic development rate may not always be positively related to lifetime fitness, since delayed effects of larval development persist also across life history stages.

Genetic diversity, but not hatching success, is jointly affected by postglacial colonization and isolation in the threatened frog, Rana latastei

Both postglacial colonization and habitat fragmentation can reduce the genetic diversity of populations, which in turn can affect fitness. However, since these processes occur at different spatial and temporal scales, the consequences of either process may differ. To disentangle the relative role of isolation and postglacial colonization in determining genetic diversity and fitness, we studied microsatellite diversity of 295 individuals from 10 populations and measured the hatch rate of 218 clutches from eight populations of a threatened frog, R. latastei. The populations that were affected by fragmentation to a greater extent suffered higher embryo mortality and reduced hatch rate, while no effects of distance from glacial refugium on hatch rate were detected. Altogether, distance from glacial refugium and isolation explained > 90% of variation in genetic diversity. We found that the genetic diversity was lowest in populations both isolated and far from the glacial refugium, and that distance from refugium seems to have the primary role in determining genetic diversity. The relationship between genetic diversity and hatch rate was not significant. However, the proportion of genetic diversity lost through recent isolation had a significant, negative effect on fitness. It is possible that selection at least partially purged the negative effects of the ancestral loss of genetic diversity.

Potential range of the invasive fish rotan (Perccottus glenii) in the Holarctic

Rotan Perccottus glenii is one of the most widespread alien invasive freshwater fish in Eurasia. We reviewed the mechanisms of its dispersion, identified the potential range and regarded these results in the light of possible prevention of further expansion. Our analysis was based on 970 presence records and 198 absence records from both invaded and native parts of the range. Since 1916 invasion dynamics of rotan have been driven by episodic anthropogenic translocations, followed by spontaneous expansion within river networks, and secondary translocations. Maxent species distribution models with independent validations showed that rotan distribution in Eurasia has climatic limitations; rotan have already invaded most areas with high climatic suitability in the Palearctic, but some regions of North Eurasia and North America, where rotan is currently absent, have high climatic suitability and may be vulnerable to invasion in the future. Rotan’s high invasiveness, lack of geographical barriers and absence of reliable methods to prevent spread present a very high risk of expansion within appropriate climate limits in Europe. Our analysis shows that the long-term invasion dynamics of an invasive freshwater species may depend on climate variables rather than on river basin borders. Species distribution models, based on large scale environmental layers, can be useful to understand the invasion risk for other freshwater species restricted to shallow aquatic habitats.

Supplementation or in situ conservation? Evidence of local adaptation in the Italian agile frog Rana latastei and consequences for the management of populations

Relocation of endangered species can be an effective conservation tool if it does not mix populations that represent significant intraspecific variation. The threatened Italian agile frog, Rana latastei, has small populations with low genetic diversity: translocation has been proposed to improve the likelihood of survival of populations. Using a common environment experiment and field surveys, we investigated whether there were differences in larval growth and developmental rate between foothill and lowland R. latastei populations, to evaluate if they are evolutionarily significant units. In nature, the colder climate of the foothills causes delayed metamorphosis. Conversely, in a common environment, larvae from foothill populations show faster growth and development. We did not find a significant egg-size related maternal effect or any differences in size at metamorphosis. We hypothesise that counter-gradient selection promoted fast growing phenotypes in a cold environment, where low temperatures slow down larval development. Foothill populations, despite being only a small geographical distance away from lowland populations, seem to be adapted to a colder climate and represent an evolutionarily significant unit. Different populations should, therefore, be managed independently, avoiding translocation. We suggest that evolutionary divergence between populations should be verified prior to planning relocation programmes, to prevent the risk of genetic homogenisation.

Landscape–stream interactions and habitat conservation for amphibians

Semiaquatic organisms depend on the features of both water bodies and landscapes; the interplay between terrestrial and aquatic systems might influence the semiaquatic communities, determining the scale at which management would be more effective. However, the consequences of such interplay are not frequently quantified, particularly at the community level. We analyzed the distribution of amphibians to evaluate whether the influence of landscape features on freshwater ecosystems can have indirect consequences at both the species and community level. We surveyed 74 streams in northern Italy to obtain data on breeding amphibians, water, and microhabitat features; we also measured features of surrounding landscapes. We used an information-theoretic approach and structural equation models to compare hypotheses on causal relationships between species distribution and variables measured at multiple levels. We also used a constrained redundancy analyses to evaluate causal relationships between multivariate descriptors of habitat features and community composition. Distribution of Salamandra salamandra was related to landscape, hydrological, and water characteristics: salamanders were more frequent in permanent streams with low phosphate concentration within natural landscapes. Water characteristics were dependent on landscape: streams in natural landscapes had less phosphates. Landscape influenced the salamander both directly and indirectly through its influence on phosphates. Community structure was determined by both landscape and water characteristics. Several species were associated with natural landscapes, and with particular water characteristics. Landscape explained a significant proportion of variability of water characteristics; therefore it probably had indirect effects on community. Upland environments play key roles for amphibians, for example, as the habitat of adults, but upland environments also have indirect effects on the aquatic life stages, mediated through their influence on water characteristics. Synergistic effects can magnify the negative consequences of landscape alteration on amphibians; landscape management can be particularly effective, as it can also improve wetland features.